Selective determination of aromatic amino acids by magnetic hydroxylated MWCNTs and MOFs based composite.

A novel magnetic solid-phase extraction (MSPE) based method was established for aromatic amino acids (AAAs) selective determination. Central to the method was the adopted extractant. An explored composite of metal organic frameworks (MOFs) and magnetic carbon nanotubes (CNTs) was employed for that, which exhibited superior adsorption affinity and selectivity to AAAs as compared to other amino acids with the mechanisms attributed to multiple hydrogen bonding and π-π electron-donor-acceptor (EDA) interactions. The morphology, structure and magnetic behavior of the composite were characterized and related MSPE procedure was established. Critical extraction conditions including pH, extraction time, temperature and salt addition were investigated and optimized. Subsequently the concentrations of three AAAs tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) in Lanzhou lily were determined by the composite based MSPE procedure coupled with high performance liquid chromatography-ultraviolet detection (HPLC-UV). The composite provided a superior sample clean-up function and many of the matrix interferences were eliminated, thus ensured AAAs were accurately and efficiently determined. The results showed that the method had good linearities with the linear coefficients above 0.99, desirable recoveries ranged from 88.0% to 96.8% with the RSD less than 5.1%, satisfactory precision and low limits of detection (LODs) which were respectively 0.04, 0.11, and 0.87ng/g for Trp, Tyr and Phe. The composite based MSPE-HPLC-UV method has great potentials for the AAAs selective determination from complex matrix samples.

Synergetic mechanism and enantioseparation of aromatic ß-amino acids by biphasic chiral high-speed counter-current chromatography.

A biphasic chiral recognition system based on chiral ligand exchange with Cu(II)-N-n-dodecyl-L-proline and hydroxypropyl-ß-cyclodextrin as an additive was developed to enantioseparate aromatic ß-amino acids by high-speed counter-current chromatography. The biphasic chiral recognition system was established with an n-butanol/water (1:1, v/v) solvent system by adding N-n-dodecyl-L-proline and Cu(II) ions to the organic phase and hydroxypropyl-ß-cyclodextrin to the aqueous phase. Several separation parameters, such as temperature, pH value, and chiral selector concentration, were systematically investigated by enantioselective liquid-liquid extraction. Under the optimal separation conditions, 54.5 mg of (R,S)-ß-phenylalanine and 74.3 mg of (R,S)-ß-3,4-dimethoxyphenylalanine were baseline enantioseparated. More importantly, the synergistic enantiorecognition mechanism, based on the Cu(II)-N-n-dodecyl-L-proline and hydroxypropyl-ß-cyclodextrin, was discussed for the first time.

Chiral ligand-exchange resolution of underivatized amino acids on a dynamically modified stationary phase for RP-HPTLC.

The synthesis of Spi(τ-dec), derived from the selective alkylation of L-spinacine (4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid) at the τ-nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ-dec) was successfully employed in the past to prepare home-made chiral columns for chiral ligand-exchange high-performance liquid chromatography. In the present article a new method is described, using Spi(τ-dec) as a chiral selector in high-performance thin-layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ-dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive.

Chiral separation-based ligand exchange by open-tubular capillary electrochromatography.

Chiral ligand-exchange enantioseparation of aliphatic and aromatic amino acids was successfully performed using a new open-tubular zwitterionic column with tentacle-type polymer stationary phase. The polymeric stationary phase was prepared using 3-chloro-2-hydroxypropyl methacrylate (HPMA-Cl), a new reactive monomer. The preparation procedure of the open-tubular column included silanization, in situ graft polymerization with HPMA-Cl, and L-histidine (L-His) modification. L-His was used as a chiral ligand-exchange selector and copper(II) as a central ion. Successful enantioseparation of six pairs of amino acid enantiomers was achieved with a buffer of 5 mM CuSO4, 20 mM (NH4)2SO4 at pH 3.0.

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector, N-methacryloyl-L-histidine methyl ester.

A new type of polymethacrylate-based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely D,L-phenylalanine, D,L-tyrosine, and D,L-tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N-methacryloyl-L-histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate-based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that L-enantiomers of aromatic amino acids migrated before D-enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 µm id and 375 µm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through-pore structure.

Enantioselective separation of chiral aromatic amino acids with surface functionalized magnetic nanoparticles.

Chiral resolution aromatic amino acids, DL-tryptophan (DL-Trp), DL-phenylalanine (DL-Phe), DL-tyrosine (DL-Tyr) from phosphate buffer solution was achieved in present study employing the concept of selective adsorption by surface functionalized magnetic nanoparticles (MNPs). Surfaces of magnetic nanoparticles were functionalized with silica and carboxymethyl-ß-cyclodextrin (CMCD) to investigate their adsorption resolution characteristics. Resolution of enantiomers from racemic mixture was quantified in terms of enantiomeric excess using chromatographic method. The MNPs selectively adsorbed L-enantiomers of DL-Trp, DL-Phe, and DL-Tyr from racemic mixture and enantiomeric excesses (e.e.) were determined as 94%, 73% and 58%, respectively. FTIR studies demonstrated that hydrophobic portion of enantiomer penetrated into hydrophobic cavity of cyclodextrin molecules to form inclusion complex. Furthermore, adsorption site was explored using XPS and it was revealed that amino group at chiral center of the amino acid molecule formed hydrogen bond with secondary hydroxyl group of CMCD molecule and favorability of hydrogen bond formation resulted in selective adsorption of L-enantiomer. Finally, stability constant (K) and Gibbs free energy change (-ΔG°) for inclusion complexation of CMCD with L-/D-enantiomers of amino acids were determined using spectroflurometry in aqueous buffer solution. Higher binding constants were obtained for inclusion complexation of CMCD with L-enantiomers compared to D-enantiomers which stimulated enantioselective properties of CMCD functionalized magnetite silica nanoparticles.

Electrophoretic separation with 2-mm inner diameter fused-silica microcolumn packed with quartz microncrystals and its application.

The feasibility of a microcolumn electrophoresis technique was investigated with a 100mm length, 2mm I.D. fused-silica microcolumn packed with uniform quartz microncrystals prepared by hydrothermal synthesis. To evaluate the separation technique, tryptophan, phenylalanine and tyrosine were primarily separated by the microcolumn electrophoresis and detected at 216 nm without derivatization by an ordinary spectrophotometer. The separation conditions of the amino acids were optimized. With 1.5 mmol/L disodium phosphate buffer solution (pH 11.5) containing 25% (v/v) methanol and 10% (v/v) acetonitrile, the three amino acids were separated and the separation efficiency of tryptophan was 4.5x10(4)plates/m. The limits of detection were 0.035, 0.22 and 0.20 micromol/L, respectively. The sample capacity of the electrophoretic microcolumn achieved 35 microL. The proposed method was used to determine these amino acids in compound amino acid injection samples without derivatization. For the simplicity and portability of the microcolumn electrophoresis, it is studied as one of the high-performance separation techniques for an in situ and real-time electrokinetic flow analysis system. For its high detection sensitivity and large sample capacity, it can be developed for preparative electrophoresis.

Coupling process between solid-liquid extraction of amino acids by calixarenes and photocatalytic degradation.

The removal of biological or pharmaceutical compounds through the wastewater treatment becomes relevant. These compounds are present as traces in sewage. In this work, we propose a coupling process which combines the pre-concentration of the pollutant by selective extraction and then degradation of these pollutants by photocatalysis in presence of TiO2. This process is efficient at room temperature by activation of a photocatalyst (TiO2) under UV light. Aromatic amino acids were chosen as model of pharmaceutical pollutants. Their extraction from water, ensured by calixarene derivatives, and their photocatalytic degradation were investigated. It was shown that photodegradation follows a first-order kinetic and that the rate constant enhances with amino acid concentration. The effect of the pH on the rate constant will be discussed.

Chiral CE of aromatic amino acids by ligand-exchange with zinc(II)-L-lysine complex.

A novel method of chiral ligand-exchange CE was developed with either L- or D-lysine (Lys) as a chiral ligand and zinc(II) as a central ion. This type of chiral complexes was explored for the first time to efficiently separate either individual pairs of or mixed aromatic amino acid enantiomers. Using a running buffer of 5 mM ammonium acetate, 100 mM boric acid, 3 mM ZnSO(4) x 7H(2)O and 6 mM L-Lys at pH 7.6, unlabeled D,L-tryptophan, D,L-phenylalanine, and D,L-tyrosine were well separated, giving a chiral resolution of up to 7.09. The best separation was obtained at a Lys-to-zinc ratio of 2:1, zinc concentration of 2-4 mM and running buffer pH 7.6. The buffer pH was determined to have a strong influence on resolution, while buffer composition and concentration impacted on both the resolution and peak shape. Boric acid with some ammonium acetate was an adoptable buffer system, and some additives like ethylene diamine tetraacetic acid capable of destroying the complex should be avoided. Fine-tuning of the chiral resolution and elution order was achieved by regulating the ratio of L-Lys to D-Lys; i.e. the resolution increased from zero to its highest value as the ratio ascended from 1:0 to 1:infinitive, and L-isomers eluted before or after D-isomers in excessive D- or L-Lys, respectively.

Comparison between bioartificial and artificial liver for the treatment of acute liver failure in pigs.

AIM: To characterize and evaluate the therapeutic efficacy of bioartificial liver (BAL) as compared to that of continuous hemodiafiltration (CHDF) with plasma exchange (PE), which is the current standard therapy for fulminant hepatic failure (FHF) in Japan. METHODS: Pigs with hepatic devascularization were divided into three groups: (1) a non-treatment group (NT; n = 4); (2) a BAL treatment group (BAL; n = 4), (3) a PE + CHDF treatment group using 1.5 L of normal porcine plasma with CHDF (PE + CHDF, n = 4). Our BAL system consisted of a hollow fiber module with 0.2 mum pores and 1 multiply 10(10) of microcarrier-attached hepatocytes inoculated into the extra-fiber space. Each treatment was initiated 4 h after hepatic devascularization. RESULTS: The pigs in the BAL and the PE + CHDF groups survived longer than those in the NT group. The elimination capacity of blood ammonia by both BAL and PE + CHDF was significantly higher than that in NT. Aromatic amino acids (AAA) were selectively eliminated by BAL, whereas both AAA and branched chain amino acids, which are beneficial for life, were eliminated by PE + CHDF. Electrolytes maintenance and acid-base balance were better in the CPE + CHDF group than that in the BAL group. CONCLUSION: Our results suggest that PE + CHDF eliminate all factors regardless of benefits, whereas BAL selectively metabolizes toxic factors such as AAA. However since PE + CHDF maintain electrolytes and acid-base balance, a combination therapy of BAL plus CPE + CHDF might be more effective for FHF.

Hybrid silica monolithic column for capillary electrochromatography with enhanced cathodic electroosmotic flow.

A hybrid silica monolithic stationary phase for RP CEC was prepared by in situ co-condensation of (3-mercaptopropyl)-trimethoxysilane (MPTMS), phenyltriethoxysilane (PTES), and tetraethoxysilane (TEOS) via a sol-gel process. The thiol groups on the surface of the stationary phase were oxidized to sulfonic acids by peroxytrifluoroacetic acid. The introduced sulfonic acid moieties on the monoliths were characterized by a strong and relatively stable EOF in a broad pH range from 2.35 to 7.0 in CEC. Aromatic acids and neutral compounds can be simultaneously separated in this column under cathodic EOF. The CEC column exhibited a typical RP chromatographic mechanism for neutral compounds due to the introduced phenyl groups.

Capillary electrophoresis direct enantioseparation of aromatic amino acids based on mixed chelate-inclusion complexation of aminoethylamino-beta-cyclodextrin.

6(A)-(2-Aminoethylamino)-6(A)-deoxy-beta-cyclodextrin (CDen) was synthesized and formed a binary complex with Cu(II) which was shown to be an effective chiral selector for separation of underivatized amino acid enantiomers in capillary electrophoresis (CE). Moreover, the chiral resolution was greatly enhanced by the presence of polyethyl glycol (PEG) and tert-butyl alcohol in the running buffer. The optimum experimental conditions were 20 mmol/L CDen, 20 mmol/L CuSO(4).5H(2)O, 5.0 mg/mL PEG20000 and 1.0% v/v tert-butyl alcohol, pH 5.80. With the proposed method, the four selected aromatic chiral amino acid pairs were separated in less than 15 min.

Enantioseparation of aromatic amino acids and amino acid esters by capillary electrophoresis with crown ether and prediction of enantiomer migration orders by a three-dimensional quantitative structure-property relationship/comparative field analysis model.

The complete enantioseparations of eight aromatic amino acids and four alkyl esters of 2-phenylglycine were achieved by chiral capillary electrophoresis employing 20 mM Tris-citric acid background electrolyte (pH 2.50) containing 5.0 mM of (+)-18-crown-6-tetracarboxylic acid. The relative migration times (RMTs) to that of 6-aminonicotinic acid were characteristic of each enantiomer with good within-day precisions (% relative standard deviation (RSD) < or = 2.0). Quantitative structure-property relationship (QSPR) modeling based on comparative molecular field analysis (CoMFA) was performed to investigate the correlation between the molecular field descriptor values of each enantiomer studied as analyte and its RMT. The resulting CoMFA model allowed reliable prediction for the RMT values (q2= 0.406, r2 = 0.996), thus being expected to become a valuable tool to predict enantiomer migration orders (EMOs) of amino acids and amines whose pure enantiomers are unavailable. The CoMFA steric fields supported the well-established chiral recognition mechanism based on molecular interaction between chiral selector (+)-18-crown-6-tetracarboxylic acid and amino acid enantiomers.

Selectivity control in the separation of aromatic amino acid enantiomers with sulphated beta-cyclodextrin.

Control of selectivity in the enantiomeric separation of three aromatic amino acids (phenylalanine, tyrosine and tryptophan) is demonstrated by electrokinetic capillary chromatography utilising temperature variations coupled with the use of sulphated-beta-cyclodextrin (s-beta-CD) as a pseudostationary phase. The concentration of s-beta-CD and temperature were used as experimental variables to control the observed selectivity. A double-coated capillary was used and proved very robust with reproducibility of migration times being <2.0% R.S.D. between runs and <2.6% on using a new capillary. The system was modelled successfully using an artificial neural network (ANN) comprising one input layer, two hidden layers and one output layer. The model accurately described the observed separations with a correlation coefficient of 0.999 being observed between predicted and observed migration times. Selectivity optimisation was achieved using the normalised resolution product and minimum resolution criteria, with both providing optima at different experimental conditions. The selectivity changes observed also allowed the estimation of electrolyte temperatures within the capillary at high operating currents (>100 microA). Using a 50 microm i.d. capillary and an electrolyte comprising 20 mM phosphate and 15 mM s-beta-CD, a temperature of 52 degrees C was calculated within the capillary at an applied voltage of +30 kV.

Optimisation of selectivity in the separation of aromatic amino acid enantiomers using sulfated beta-cyclodextrin and dextran sulfate as pseudostationary phases.

Control of selectivity in the enantiomeric separation of three aromatic amino acids (phenylalanine, tyrosine and tryptophan) was demonstrated utilising two separate electrolyte additives. Sulfated-beta-cyclodextrin (s-beta-CD) was chosen as the chiral selector while the addition of dextran sulfate provided a means with which to predictably fine-tune separation selectivity. The two additives were found to interact independently with the amino acids, with the s-CD providing chiral interactions while the dextran sulfate provided ion-exchange (IE) interactions. The system was also very robust with reproducibility of migration times being < 2.0% RSD between runs and < 2.6% on using a new capillary. A physical model derived from first principles was also successfully used to describe the two additive system. The model accurately described the observed separations over the range of 0-20 mM s-beta-CD and 0-1% dextran sulfate with a correlation coefficient of 0.998 between predicted and observed mobilities. The physical model also provided useful information about the system including association constants between the analytes and the pseudostationary phases, together with the mobilities of the associated complexes (analyte-cyclodextrin and analyte-dextran sulfate). Selectivity optimisation was achieved using the normalised resolution product and minimum resolution criteria. The physical model also allowed a desired separation selectivity to be obtained, such that experimental conditions could be predicted to lead to a particular migration order.

Enantioseparation of chiral aromatic amino acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes.

Simultaneous enantioseparations of 15 racemic aromatic amino acids and L-mimosine for their chiral discrimination were achieved by neutral selector-modified capillary electrophoresis (CE) and by charged selector-modified CE. Among the diverse cyclodextrins (CDs) examined, hydroxypropyl (HP)-alpha-CD as the neutral selector and highly sulfated (HS)-gamma-CD as the charged selector provided best chiral environments of different enantioselectivities. Fairly good enantiomeric resolutions were achieved with the HP-alpha-CD mode except for racemic 6-hydroxy-3,4-dihydroxyphenylalanine, threo-3,4-dihydroxyphenylserine and homophenylalanine while high-resolution separations of all the enantiomeric pairs were achieved in the HS-gamma-CD mode except that L-mimosine was not detected and a partial resolution (0.6) for threo-3,4-dihydroxyphenylserine enantiomers. Relative migration times to that of internal standard under the respective optimum conditions were characteristic of each enantiomer with good precision (% RSD: 0.7-3.8), thereby enabling to cross-check the chemical identification of aromatic amino acids and also their chiralities. The method linearity was found to be adequate (r> 0.99) for the chiral assay of the aromatic amino acids investigated. When applied to extracts of three plant seeds, nonprotein amino acids such as L-mimosine (42 nug/g) from Mimosa pudica Linné, and L-3,4-dihydroxyphenylalanine (268 nug/g) from Vicia faba were positively detected along with L-tryptophan, L-phenylalanine and L-tyrosine.